Moveable chromatography column separator

ABSTRACT

The current invention is directed to a chromatography column separator which separates the chromatography column in an upper chromatography column chamber and a lower chromatography column chamber, and which has a variable position within the chromatography column, and which is embedded by the chromatography material. The separator allows the replacement of the chromatography material in the upper chromatography column chamber without the need to replace the chromatography column material in the lower chromatography column chamber and it allows also the combination of two different chromatography materials with different chromatographical functional groups in one chromatography column.

This application is a divisional of U.S. patent application Ser. No.13/386,035, filed Jan. 20, 2012, allowed, which is a § 371 ofInternational Application No. PCT/EP2010/004621 filed Jul. 28, 2010, andclaims priority from European Patent Application No. 09009859.1 filedJul. 30, 2009, each of which is incorporated herein by reference in itsentirety for all purposes.

The current invention is in the field of chromatography, in more detailin the field of polypeptide purification by chromatographic methodsemploying chromatography columns. Herein is reported a chromatographycolumn separator which can divide a chromatography column in an upperchromatography column chamber and a lower chromatography column chamber,and which has a variable position within the chromatography column, i.e.the separator can slide along with the pressure dependent compression ofthe surrounding chromatography material.

BACKGROUND OF THE INVENTION

Polypeptides play an important role in today's medical portfolio. Forhuman application every pharmaceutical substance has to meet distinctcriteria. To ensure the safety of biopharmaceutical agents to humansnucleic acids, viruses, and host cell proteins, which would cause severeharm, have to be removed especially. To meet the regulatoryspecification one or more purification steps have to follow themanufacturing process. Among other purity, throughput, and yield play animportant role in determining an appropriate purification process.

In general chromatographic methods chromatography columns are employedwhich are essentially comprising a column housing with an upper andlower fitting which in turn comprises an inlet at the top of the column,an outlet at the bottom of the column, a upper chromatography columnfrit, a lower chromatography column frit, an upper distributor plate anda chromatographic material.

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5^(th) edition,Part A: Fundamentals and Techniques, Heftmann, E. (eds), ElsevierScience Publishing Company, New York, (1992); Advanced Chromatographicand Electromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, Protein Purification: Principles and Practice(1982); Sambrook, J., et al. (eds), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel,F. M., et al. (eds), John Wiley & Sons, Inc., New York.

For the purification of immunoglobulins, which have been produced e.g.by cell cultivation methods, in general a combination of differentchromatography steps is employed. Normally a protein A affinitychromatography is followed by one or two additional separation steps.The final purification step is a so called “polishing step” for theremoval of trace impurities and contaminants like aggregatedimmunoglobulins, residual HCP (host cell protein), DNA (host cellnucleic acid), viruses, or endotoxins. For this polishing step normallyan anion exchange material in a flow-through mode is used.

In WO 2006/048514 a container comprising open, complementarily-shapedupper and lower ends with a chromatographic mixture which is placed inthe container, and a membrane filter which is fixed to the lower end ofthe container is reported. A high throughput liquid chromatographycolumn assembly including a loading column with a loading chamber with afirst inner diameter and a first length in fluid communication with aseparation column having a separation chamber with a diameter smallerthan the loading column's inner diameter and a length greater than theloading column's length is reported in U.S. Pat. No. 6,458,273.

High pressure liquid chromatography columns are reported in U.S. Pat.No. 4,719,011 which may be modularly modified as to length and/orinternal diameter and may contain other components in the modularsystem, for example, column sections, adapters and cone adapters, forjoining column sections of different internal diameters, and end plateunits for funneling in or discharging out fluids. A column devicecomprising a stationary phase having a plurality of particles adaptedfor interacting with a mobile phase in order to separate differentcompounds of a sample fluid dissolved in the mobile phase, a housing forat least partly housing the stationary phase, and a separator separatingsections of the stationary phase and being force-coupled with thehousing is reported in EP 1 916 522.

A segmented chromatography column including a plurality of media bedsseparated and bounded by a plurality of porous members is reported in WO00/010675. In U.S. Pat. No. 3,398,512 a chromatography apparatus isreported. Chromatography columns with partition elements therein arereported in U.S. Pat. No. 3,453,811. In EP-A 1 892 526 a column andcartridge column using the same is reported. A separation system for theresolving of volatile mixtures is reported in U.S. Pat. No. 3,657,864.

SUMMARY OF THE INVENTION

This separator as reported herein allows the replacement of thechromatography material in the upper chromatography column chamberwithout the need to replace the chromatography column material in thelower chromatography column chamber, it also allows e.g. the combinationof two different chromatography materials with differentchromatographical functional groups in one chromatography column, and inthe presence of the separator more homogeneously packed chromatographycolumns can be provided. Further the separator as reported hereinreduces the backpressure exerted by the column, i.e. it reduces thepressure inside the column. At each separator added to the columnpacking a pressure drop is induced.

Herein is reported as one aspect a guide ring of circular shape for usein a liquid chromatography column, characterized in that the guide ringhas a vertical cross-section comprising two axially symmetriccross-section areas (5 and 6), wherein each of the axially symmetriccross-section areas has

-   -   a) a tapering structure, wherein the tapering is from the        outside to the inside of the guide ring, and    -   b) a notch (8) with an opening directed to the inside of the        guide ring for mounting a frit.

In one embodiment the notch is a rectangular notch. In a furtherembodiment each of the cross-section areas has a triangular shape andthe longest side (7) has a length of at least 1.5 times the diameter ofthe notch (8). In a further embodiment the ring is made of rubber,plastic, silicone, polytetrafluoroethylene, polyethylene, orpolypropylene.

Also an aspect as reported herein is the use of a guide ring as reportedherein for mounting a frit into a cylindrical liquid chromatographycolumn.

Also an aspect is a chromatography column separator comprising a guidering as reported herein and a frit mounted into the guide ring.

In one embodiment the frit is

-   -   a) a single frit, or    -   b) two frits with an upper frit and a lower frit.

In another embodiment a) the frit has a pore size of from 1 μm to 20 μm,or b) each of the frits has a pore size of from 1 μm to 20 μmindependently of each other whereby the pore size of the upper frit issmaller than the pore size of the lower frit. In another embodiment thefrit is made of metal, silicone, polypropylene, polyethylene,polytetrafluoroethylene, sintered materials or combinations thereof. Ina further embodiment the separator comprises distance holders allattached to one side of the separator.

In one embodiment the chromatography column separator is characterizedin that

-   -   a) the separator separates a chromatography column in an upper        chromatography column chamber and a lower chromatography column        chamber, and    -   b) the separator has a variable position within the        chromatography column by sliding along the inner wall of the        chromatography column.

In one embodiment the chromatography column separator comprises onefrit, in another embodiment the separator comprises an upper frit and alower frit. In a further embodiment the chromatography column separatoror said upper frit or said lower frit has a pore size of from 1 μm to 20μm, whereby the pore size of the upper frit is smaller than the poresize of the lower frit. In another embodiment the frit is made of metal,silicone, polypropylene, polyethylene, polytetrafluoroethylene, sinteredmaterials or combinations thereof. In one embodiment the chromatographycolumn separator has three or more distance holders attached to oneside. In a specific embodiment the distance holder are attached to theside of the chromatography column separator with the larger pore size.

Another aspect as reported herein is the use of a chromatography columnseparator as reported herein for dividing a chromatography column inchambers. A further aspect as reported herein is the use of achromatography column separator as reported herein for separating achromatography column in an upper chromatography column chamber and alower chromatography column chamber. Still another aspect is the use ofa chromatography column separator as reported herein for separating thechromatography material in a chromatography column in two distinctfractions. Also an aspect as reported herein is the use of achromatography column separator as reported herein for separating twodifferent chromatography materials in one chromatography column.

Further aspects as reported herein are the use of a chromatographycolumn separator according to the invention for separating achromatography column containing a chromatography material in twoseparate chambers, whereby the chromatography column separator isembedded within a chromatography material in a chromatography column,the use of a chromatography column separator as reported herein forseparating a chromatography column containing a chromatography materialin two separate chambers, whereby the chromatography column separatorseparates two different chromatography materials in one chromatographycolumn.

Still a further aspect as reported herein is a chromatography columncomprising at least one, i.e. one or more, chromatography columnseparator as reported herein. In one embodiment the chromatographycolumn comprises one or two chromatography column separators. In afurther embodiment of this aspect any chromatography column separator isin contact with a first chromatography material and in contact with asecond chromatography material, whereby the first and the secondchromatography material are

-   -   a) chromatography materials with the same chromatographical        functional group and of the same or different particle size, or    -   b) chromatography materials with different chromatographical        functional groups.

Another aspect as reported herein is the use of a chromatography columncomprising at least one chromatography column separator as reportedherein for the purification of a polypeptide.

Also an aspect as reported herein is a chromatography column separatorapplication device, whereby the device has at its bottom a shape that isinverse to the shape of the upper surface of the chromatography columnseparator as reported herein and a diameter that is smaller than theouter diameter of the guide ring.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a guide ring of circular shape for use in a liquidchromatography column, characterized in that the guide ring has avertical cross-section comprising two axially symmetric cross-sectionareas, wherein each of the axially symmetric cross-section areas has

-   -   a) a tapering structure, wherein the tapering is from the        outside to the inside of the guide ring, and    -   b) a notch with an opening directed to the inside of the guide        ring for mounting a frit.

It is reported a chromatography column separator for separating achromatography material in a chromatography column in two separatedparts, whereby

-   -   a) the separator separates the chromatography column in an upper        chromatography column chamber and a lower chromatography column        chamber, and    -   b) the separator has a variable position within the        chromatography column, and    -   c) the separator is embedded in the chromatography material.

In column chromatography at least one or more polypeptides of interest,i.e. one or more polypeptides to be purified, are separated from otherpolypeptides and substances not of interest by a chromatographicseparation method via the interaction with a chromatography material.

The term “polypeptide” denotes a polymer consisting of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically. Polypeptides of less than about 20 amino acid residuescan be referred to as “peptides”. A polypeptide comprising one or moreamino acid chains or one amino acid chain of 100 or more amino acidresidues can be referred to as “protein”. Polypeptides additionally mayalso comprise non-amino acid components, such as carbohydrate groups.Carbohydrate groups and other non-amino acid components may be added bythe cell in which the polypeptide is produced, and will vary with thetype of cell. Polypeptides are defined in terms of their amino acidbackbone structures; substituents such as carbohydrate groups aregenerally not specified, but may be present nonetheless. In oneembodiment the polypeptide is an immunoglobulin, or an immunoglobulinfragment, or a fusion protein comprising an immunoglobulin or animmunoglobulin fragment.

The term “immunoglobulin” denotes a polypeptide comprising in generaltwo light chain polypeptides (light chain, LC) and two heavy chainpolypeptides (heavy chain, HC). Each of the heavy and light chainscomprises a variable region (generally the amino terminal portion of thechain) which contains specific binding regions (CDR, complementarydetermining region) which interact with the antigen. Each of the heavyand light chains also comprises a constant region (generally, thecarboxyl terminal portion of the chains) which mediate the binding ofthe immunoglobulin to host tissues or factors including various cells ofthe immune system, some phagocytic cells and a first component (C1q) ofthe classical complement system. In general a light chain comprises alight chain variable domain and a light chain constant domain, whereas aheavy chain comprises a heavy chain variable domain, a hinge region, andheavy chain constant domains, i.e. a C_(H)1 domain, a C_(H)2 domain, aC_(H)3 domain, and optionally a C_(H)4 domain. Immunoglobulins may existin a variety of forms, including, for example, the fragments Fv, Fab,and F(ab)₂ as well as single chains (e.g. Huston, J. S., et al., Proc.Natl. Acad. Sci. USA 85 (1988) 5879-5883; Bird, R. E., et al., Science242 (1988) 423-426; in general, Hood, L. E., et al., Immunology,Benjamin N. Y., 2nd edition (1984) and Hunkapiller, T., and Hood, L.,Nature 323 (1986) 15-16). Depending on the amino acid sequence of theconstant domains of the heavy chain immunoglobulins are assigned todifferent classes: IgA, IgD, IgE, IgG, and IgM. Some of these classesare further divided into subclasses (isotypes), e.g. IgG in IgG1, IgG2,IgG3, and IgG4, or IgA in IgA1 and IgA2. According to the immunoglobulinclass to which an immunoglobulin belongs the heavy chain constantregions of immunoglobulins are called a (IgA), d (IgD), e (IgE), g(IgG), and m (IgM), respectively. In addition two types ofimmunoglobulin light chain are possible, a lambda type light chain and akappa type light chain.

The term “chromatography material” as used within this applicationdenotes on the one hand a solid material that can be used withoutfurther modification in a chromatographic purification of a polypeptideof interest, such as hydroxyapatite, and also material comprising a bulkcore material which has been modified by the introduction/coupling ofchromatographical functional groups, for example by covalent bonds, suchas SP-Sepharose®. The bulk core material is understood to be notinvolved in the chromatographic separation, i.e. in the interactionbetween the polypeptide to be separated and the chromatographicalfunctional groups of the chromatography material. It is merely providinga three dimensional framework to which the chromatographical functionalgroups are attached and which ensures that the solution containing thepolypeptide to be purified can access the chromatographical functionalgroups. In one embodiment the bulk core material is a solid phase. Thus,in another embodiment the “chromatography material” is a solid phase towhich chromatographical functional groups are attached. In anotherembodiment the “chromatographical functional group” is an ionizablehydrophobic group, or a hydrophobic group, or a complex group in whichdifferent chromatographical functional groups are combined in order tobind only a certain type of polypeptide, or a covalently boundpositively or negatively charged group.

Generally the polypeptide to be purified is applied to thechromatography material as an aqueous, buffered solution.

The term “applying to” and grammatical equivalents thereof as usedwithin this application denotes a partial step of a chromatographicpurification of a polypeptide wherein a solution containing thepolypeptide of interest to be purified is brought in contact with thechromatography material. This denotes that the solution containing thepolypeptide to be purified is added to the chromatography columncontaining the chromatography material at the upper inlet of the column.The solution containing the polypeptide of interest to be purifiedpasses through the chromatography material thereby allowing for aninteraction between the chromatography material and the substances insolution. Depending on the conditions, such as e.g. pH, conductivity,salt concentration, temperature, and/or flow rate, substances containedin the solution specifically interact with the chromatography material,whereby their movement through the chromatography column is effecteddepending on the interaction with the chromatography material. Thepolypeptide of interest can be recovered from the solution obtainedafter the purification step, i.e. from the eluate, by methods familiarto a person of skill in the art, such as e.g. precipitation, saltingout, ultrafiltration, diafiltration, lyophilization, affinitychromatography, or solvent volume reduction to obtain the substance ofinterest in substantially homogeneous form.

The term “buffered” as used within this application denotes a solutionin which changes of pH due to the addition or release of acidic or basicsubstances is leveled by a buffer substance. Any buffer substanceresulting in such an effect can be used. In one embodimentpharmaceutically acceptable buffer substances are used, such as e.g.phosphoric acid or salts thereof, acetic acid or salts thereof, citricacid or salts thereof, morpholine or salts thereof, 2-(N-morpholino)ethanesulfonic acid or salts thereof, histidine or salts thereof,glycine or salts thereof, or Tris (hydroxymethyl) aminomethane (TRIS) orsalts thereof. In one embodiment the buffer substance is phosphoric acidor salts thereof, or acetic acid or salts thereof, or citric acid orsalts thereof, or histidine or salts thereof. Optionally the bufferedsolution may comprise an additional salt, such as e.g. sodium chloride,sodium sulphate, potassium chloride, potassium sulfate, sodium citrate,or potassium citrate.

In a liquid column chromatography purification method the chromatographymaterial is located within a column housing and denoted as “stationaryphase”. To enable a stationary phase to interact withsubstances/polypeptides in a solution applied to it, the stationaryphase is surrounded by/embedded in a “mobile phase”. The term “mobilephase” denotes a liquid, e.g. a buffered, aqueous solution, a mixture ofwater and an organic solvent, or an organic solvent, which is used inthe chromatographic purification method in which a stationary phase isemployed.

Different chromatography methods are well established and widespreadused for polypeptide recovery and purification, such as affinitychromatography with microbial proteins (e.g. protein A or protein Gaffinity chromatography), ion exchange chromatography (e.g. cationexchange (carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis) (see e.g.Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

The term “hydrophobic charge induction chromatography”, short “HCIC”,denotes a chromatography method which employs a “hydrophobic chargeinduction chromatography material”. A “hydrophobic charge inductionchromatography material” is a chromatography material which compriseschromatographical function groups which can in one pH range formhydrophobic bonds to the substance/polypeptide to be purified and whichare charged either positively or negatively in other pH ranges, i.e.HCIC uses ionizable hydrophobic groups as chromatographical functionalgroup. Generally the polypeptide is bound to the hydrophobic chargeinduction material under neutral pH conditions and recovered afterwardsby the generation of charge repulsion by a change of the pH value. Anexemplary “hydrophobic charge induction chromatography materials” isBioSepra MEP or HEA Hypercel (Pall Corp., USA).

The term “hydrophobic interaction chromatography”, short “HIC”, denotesa chromatography method in which a “hydrophobic interactionchromatography material” is employed. A “hydrophobic interactionchromatography material” is a chromatography material to whichhydrophobic groups, such as butyl-, octyl-, or phenyl-groups, are boundas chromatographical functional groups. Applied to polypeptides areseparated depending on the hydrophobicity of their surface exposed aminoacid side chains, which can interact with the hydrophobic groups of thehydrophobic interaction chromatography material. The interactionsbetween polypeptides and the chromatography material can be influencedby temperature, solvent, and ionic strength of the solvent. Atemperature increase e.g. supports the interaction between thepolypeptide and the hydrophobic interaction chromatography material asthe motion of the amino acid side chains increases and hydrophobic aminoacid side chains buried inside the polypeptide at lower temperaturesbecome accessible. Also is the hydrophobic interaction promoted bykosmotropic salts and decreased by chaotropic salts. “Hydrophobicinteraction chromatography materials” are e.g. Phenylsepharose CL-4B, 6FF, HP, Phenyl Superose, Octyl-Sepharose CL-4B, 4 FF, and Butylsepharose4 FF (all available from Amersham Pharmacia Biotech Europe GmbH,Germany), which are obtained via glycidyl-ether coupling to the bulkmaterial.

The term “affinity chromatography” as used within this applicationdenotes a chromatography method which employs an “affinitychromatography material”. In an affinity chromatography the polypeptidesare separated based on their biological activity or chemical structuredepending on the formation of electrostatic interactions, hydrophobicbonds, and/or hydrogen bond formation to the chromatographicalfunctional groups of the chromatography material. To recover thespecifically bound polypeptide from the affinity chromatography materialeither a competitor ligand is added or the chromatography conditions,such as pH value, polarity or ionic strength of the buffer are changed.An “affinity chromatography material” is a chromatography material whichcomprises a complex chromatographical functional group in whichdifferent single chromatographical functional groups are combined inorder to bind only a certain type of polypeptide. This chromatographymaterial specifically binds a certain type of polypeptide depending onthe specifity of its chromatographical functional group. Exemplary“affinity chromatographical materials” are a “metal chelatingchromatography material” such as Ni(II)-NTA or Cu(II)-NTA containingmaterials, for the binding of fusion polypeptides containing ahexahistidine tag or polypeptides with a multitude of surface exposedhistidine, cysteine, and/or tryptophan residues, or an “antibody bindingchromatography material” such as protein A, or an “enzyme bindingchromatography material” such as chromatography materials comprisingenzyme substrate analogues, enzyme cofactors, or enzyme inhibitors aschromatographical functional group, or a “lectin binding chromatographymaterial” such as chromatography materials comprising polysaccharides,cell surface receptors, glycoproteins, or intact cells aschromatographical functional group.

The term “metal chelating chromatography” as used within thisapplication denotes a chromatography method which employs a “metalchelating chromatography material”. Metal chelating chromatography isbased on the formation of chelates between a metal ion, such as Cu(II),Ni(II) or Zn(II), which is bound to a bulk core material aschromatographical functional groups, and electron donor groups ofsurface exposed amino acid side chains of polypeptides, especially withimidazole containing side chains and thiol group containing side chains.The chelate is formed at pH values at which those side chains are atleast partly not protonated. The bound polypeptide is recovered from thechromatography material by a change in the pH value, i.e. byprotonation. Exemplary “metal chelating chromatography materials” areHiTrap Chelating HP (Amersham Pharmacia Biotec Europe GmbH, Germany), orFractogel EMD (EMD Chemicals Inc, USA).

The term “ion exchange chromatography” as used within this applicationdenotes a chromatography method which employs an “ion exchangechromatography material”. The term “ion exchange chromatographymaterial” encompasses depending whether a cation is exchanged in a“cation exchange chromatography” a “cation exchange chromatographymaterial” or an anion is exchanged in an “anion exchange chromatography”an “anion exchange chromatography material”. The term “ion exchangechromatography material” as used within this application denotes animmobile high molecular weight solid phase that carries covalently boundcharged groups as chromatographical functional groups. For overallcharge neutrality not covalently bound counter ions are associated withthe covalently bound charged groups. The “ion exchange chromatographymaterial” has the ability to exchange its not covalently bound counterions for similarly charged ions of the surrounding solution. Dependingon the charge of its exchangeable counter ions the “ion exchangechromatography material” is referred to as “cation exchangechromatography material” or as “anion exchange chromatography material”.Further depending on the nature of the charged group the “ion exchangechromatography material” is referred to as e.g. cation exchangechromatography materials with sulfonic acid groups (S), or carboxymethylgroups (CM). Depending on the chemical nature of the charged group the“ion exchange chromatography material” can additionally be classified asstrong or weak ion exchange chromatography material, depending on thestrength of the covalently bound charged substituent. For example,strong cation exchange chromatography materials have a sulfonic acidgroup as chromatographical functional group and weak cation exchangechromatography materials have a carboxylic acid group aschromatographical functional group. “Cation exchange chromatographymaterials”, for example, are available under different names from amultitude of companies such as e.g. Bio-Rex, Macro-Prep CM (availablefrom Biorad Laboratories, Hercules, Calif., USA), weak cation exchangerWCX 2 (available from Ciphergen, Fremont, Calif., USA), Dowex® MAC-3(available from Dow chemical company—liquid separations, Midland, Mich.,USA), Mustang C (available from Pall Corporation, East Hills, N.Y.,USA), Cellulose CM-23, CM-32, CM-52, hyper-D, and partisphere (availablefrom Whatrnan plc, Brentford, UK), Amberlite® IRC 76, IRC 747, IRC 748,GT 73 (available from Tosoh Bioscience GmbH, Stuttgart, Germany), CM1500, CM 3000 (available from BioChrom Labs, Terre Haute, Ind., USA),and CM-Sepharose™ Fast Flow (available from GE Healthcare—AmershamBiosciences Europe GmbH, Freiburg, Germany).

The term “hydroxyapatite chromatography” as used within this applicationdenotes a chromatography method that employs a certain form of calciumphosphate as chromatography material. Exemplary hydroxyapatitechromatography materials are Bio-Gel HT, Bio-Gel HTP, Macro-Prep Ceramic(available from Biorad Laboratories), Hydroxyapatite Type I, Type II, HAUltrogel (Sigma Aldrich Chemical Corp., USA), Hydroxyapatite Fast Flowand High Resolution (Calbiochem), or TSK gel HA-1000 (Tosoh Haas Corp.,USA).

The term “bind-and-elute mode” and grammatical equivalents thereof asused in the current invention denotes an operation mode of achromatography method, in which a solution containing a polypeptide tobe purified is applied to a chromatography material, whereby thesubstance/polypeptide to be purified binds to the chromatographymaterial. As a result the substance/polypeptide to be purified isretained on the chromatography material whereas substances/polypeptidesnot of interest are removed with the flow-through. Thesubstance/polypeptide to be purified is afterwards eluted from thechromatography material in a second step and thereby recovered from thestationary phase with an elution solution. This does not necessarilydenote that 100% of the substances/polypeptides not of interest areremoved but essentially 100% of the substances/polypeptides not ofinterest are removed, i.e. in one embodiment at least 50% of thesubstances/polypeptides not of interest are removed, in anotherembodiment at least 75% of the substances/polypeptides not of interestare removed, in a further embodiment at least 90% of thesubstances/polypeptides not of interest are removed, and in oneembodiment more than 95% of the substances/polypeptides not of interestare removed.

The term “flow-through mode” and grammatical equivalents thereof as usedwithin the current invention denotes an operation mode of achromatography method, in which a solution containing asubstance/polypeptide to be purified is applied to a chromatographymaterial, whereby the substance/polypeptide to be purified does not bindto the chromatography material. As a result the substance/polypeptide tobe purified is obtained in the flow-through. Substances/polypeptides notof interest, which were also present in the solution, bind to thechromatography material and are removed from the solution. This does notnecessarily denote that 100% of the substances/polypeptides not ofinterest are removed from the solution but essentially 100% of thesubstances/polypeptides not of interest are removed, i.e. in oneembodiment at least 50% of the substances/polypeptides not of interestare removed from the solution, in another embodiment at least 75% of thesubstances/polypeptides not of interest are removed from the solution,in a further embodiment at least 90% of the substances/polypeptides notof interest are removed from the solution, and in one embodiment morethan 95% of the substances/polypeptides not of interest are removed fromthe solution.

The terms “continuous elution” and “continuous elution method”, whichare used interchangeably within this application, denote achromatography method wherein e.g. the concentration of a substancecausing elution, i.e. the dissolution of a bound substance/polypeptidefrom a chromatography material, is raised or lowered continuously, i.e.the concentration is changed by a sequence of small steps each notbigger than a change of in one embodiment 2%, in another embodiment of1%, of the concentration of the substance causing elution. In this“continuous elution” one or more conditions, for example the pH, theionic strength, concentration of a salt, and/or the flow of achromatography method, may be changed linearly, or changedexponentially, or changed asymptotically. In one embodiment the changeis linear.

The terms “step elution” and “step elution method”, which are usedinterchangeably within this application, denote a chromatography methodwherein e.g. the concentration of a substance causing elution, i.e. thedissolution of a bound substance/polypeptide from a chromatographymaterial, is raised or lowered at once, i.e. in one embodiment directlyfrom one value/level to the next value/level. In this “step elution” oneor more conditions, for example the pH, the ionic strength,concentration of a salt, and/or the flow of a chromatography method,is/are changed all at once from a first, e.g. starting, value to asecond, e.g. final, value. The change in the step is in one embodimentbigger than a change of 5%, in another embodiment bigger than a changeof 10%, of the concentration of the substance causing elution. “Stepelution” denotes that the conditions are changed incrementally, i.e.stepwise, in contrast to a linear change. In the “step elution method”after each increase a new fraction is collected. After each increase theconditions are maintained till the next step in the elution method.

One aspect as reported herein is a chromatography column separator foruse in a chromatography column. The presence of one separator dividesthe chromatography column in an upper chromatography column chamber anda lower chromatography column chamber, and the separator has a variableposition within the chromatography column provided for by the guidering, i.e. it can slide vertically within the column depending on thepressure-dependent compression and expansion of the chromatographymaterial, and the separator is embedded in the chromatography material.

In a column chromatography separation or purification of a crudepolypeptide normally a chromatography column comprising a chromatographymaterial and a mobile phase is employed. The mobile phase is forcedthrough the chromatography column and therewith through thechromatography material by applying pressure to the mobile phase.Mediated by the mobile phase the pressure is also applied to thechromatography material whereby a pressure drop from the inlet of thechromatography column to the outlet of the chromatography column isestablished. At the outlet of the chromatography column the pressure hasdropped to the outside atmospheric pressure. Thus, to the upper fractionof the chromatography material in the chromatography column the highestpressure force is applied.

The applied pressure normally depends one the one hand on the particlesize of the chromatography material as well as on the viscosity of themobile phase as a constant flow through the chromatography column is setbut not a constant pressure. Generally the pressure increases withdecreasing chromatography material particle size. At a constant flowrate through the chromatography material a change in viscosity of themobile phase, e.g. during the regeneration or cleaning of thechromatography material, results in a change of the pressure applied tothe chromatography material. The chromatography material in general isnot a pressure insensitive material, i.e. it can be compressed andexpands after a compression. Therefore, with an increase of the appliedpressure the chromatography material is compressed and the height of thechromatography material inside the column, i.e. the bed height, isreduced. Likewise with a decrease of the applied pressure thechromatography material expands again and the height of thechromatography material inside the column increases at most to theheight before the application of the pressure. This compression andexpansion of the chromatography material is at the same time amacroscopic process of the entire chromatography material and amicroscopic process of the individual particles of the chromatographymaterial. With increasing numbers of such compression-expansion-cyclesthe particles of the chromatography material break down into smallerparticles. With decreasing particle size of the particles of thechromatography material the chromatography material packing gets morecompact and, therewith, at the same time the pressure required formaintaining a constant liquid phase flow through the column, i.e. thechromatography material, increases. This in turn again results in afurther break down of the chromatography material particles resultingagain in an increased pressure and so on.

A chromatography column separation generally can be operated up to amaximum pressure. When this upper pressure limit is reached thechromatography column packing has to be replaced in its entirety.

It has been found that a chromatography column separator as reportedherein consisting of a guide ring and a frit mounted therein does notinterfere with the chromatographical separation process but enables thatonly a fraction of the chromatography material can be exchanged, e.g.when the maximum operating pressure is reached, without the need toreplace the entire chromatography column packing. That is, thechromatography column separator according to the current inventionpermits the chromatography material in the upper chromatography columnchamber to be exchanged without interfering with the chromatographymaterial in the lower chromatography column chamber. Thus, it ispossible to remove a limited fraction of the entire chromatographymaterial contained in a chromatography column containing achromatography column separator as reported herein, e.g. after thisfraction has collapsed or is torn up or the packing quality is reduced,without the need to also remove the other fraction of the chromatographymaterial. This partial removal of the chromatography material ispossible as the separator on the one hand divides the totalchromatography material in the chromatography column in distinctfractions and on the other hand prevents the packed chromatographymaterial in the lower chromatography column chamber from being perturbedupon the removal of the chromatography material in the upperchromatography column chamber. Thus, at least the fraction of thechromatography material that is not exposed to the maximum pressurechanges and, thus, is not torn up can be used further without a negativeimpact on the separation efficiency. But, by retaining a fraction of thechromatography material a cost of goods reduction can be achieved.

The chromatography column separator as reported herein comprises a guidering into which a frit made of any inert material can be mounted. An“inert material” is a material that does not interfere with thechromatography separation process, i.e. a chromatogram obtained with achromatography column containing one or more chromatography columnseparators as reported herein is identical to a chromatogram obtainedwith a chromatography column containing no chromatography columnseparators under/with otherwise identical conditions. Such inertmaterials are e.g. metal, especially stainless steel, silicone,polypropylene, polyethylene, polytetrafluoroethylene, sintered materialsor combinations thereof, especially polytetrafluoroethylene coatedstainless steel.

The chromatography column separator as reported herein can comprise inone embodiment a single frit or in another embodiment a combination ofan upper frit and a lower frit. If the separator comprises one frit thefrit has a pore size that is smaller than the size of the particles ofthe chromatography material. In one embodiment the pore size is 10% orless of the average diameter of the particles of the chromatographymaterial in order to prevent the blocking of the pores of the frit bybroken down chromatography material particles. If the separatorcomprises two flits both frits can have the same pore size or both fritscan have different pore sizes. As the chromatography material breaksdown into smaller particles in one embodiment the upper frit has a poresize smaller than the lower frit, in one embodiment the upper frit has apore size of 10% or less of the average diameter of the particles of thechromatography material. In one embodiment the upper frit and/or thelower frit of the chromatography column separator is a metal mesh. In afurther embodiment the upper frit has a pore size smaller than the poresize of the lower frit. In one embodiment the chromatography columnseparator or the lower frit of the chromatography column separator has apore size of from 1 μm to 20 μm, or of from 1 μm to 10 μm, or of from 1μm to 5 μm. In another embodiment the upper frit of the chromatographycolumn separator has a pore size that is 5% or less of the pore size ofthe lower frit of the chromatography column separator, or the upper fritof the chromatography column separator has a pore size that is 10% ofthe pore size of the lower frit of the chromatography column separator,or the upper frit of the chromatography column separator has a pore sizethat is 20% or less of the pore size of the lower frit of thechromatography column separator. In a further embodiment the lower fritof the chromatography column separator has a pore size of 20 μm or 10 μmor 5 μm and the upper frit has a pore size of 5 μm or 1 μm.

Therefore, the use of the chromatography column separator according tothe invention allows for a longer use of the chromatography material inthe lower chromatography column chamber and concurrently for a reductionof the costs of the chromatography method.

In one embodiment the upper chromatography column chamber has of from 5%to 15%, in another embodiment 10%, of the volume of the lowerchromatography column chamber. In another embodiment the upperchromatography column chamber has of from 20% to 30%, in one embodiment25%, of the volume of the lower chromatography column chamber. In stilla further embodiment the upper chromatography column chamber has thesame volume as the lower chromatography column chamber.

It has to be pointed out that a chromatography column separator asreported herein does not make the separators or flits present at theinlet and the outlet of a chromatography column obsolete. It isfurthermore an additional separator placed inside the chromatographymaterial. As the cross-section of column chromatography columnsperpendicular to the flow direction of the mobile phase are circular thefrit and likewise the separator also has a circular outer shape. Theseparator has a height that is small compared to the overall length ofthe chromatography column packing. In one embodiment the separator has aheight of from 0.1 cm to 10 cm, in another embodiment of from 0.25 cm to5 cm, and in a further embodiment of from 0.5 cm to 2 cm. The height ofthe separator is the distance between the surface of the frit directedto the upper chromatography column chamber and the surface of the fritdirected to the lower chromatography column chamber. If the separatorcomprises an upper frit and a lower frit the combined height of the twoindividual frits may be less then the height of the separator. In thisembodiment an additional space inside the separator is present which canbe used, e.g. for placing sensors in the frit for recording e.g. the UVabsorption, or the pressure, or the conductivity of the mobile phasepassing through the separator or for providing additional valves forremoving the mobile phase or for adding a mobile phase at a positionwithin the chromatography column packing. If the separator comprises anupper frit and a lower frit both frits have in one embodiment the sameheight. In another embodiment the upper frit and the lower frit havedifferent heights.

In one embodiment the chromatography column separator has a diameterthat is smaller than the inner diameter of the chromatography column inwhich it is introduced.

In FIG. 1 exemplary chromatography column separators are shown. In FIG.1a ) a separator with a single frit is depicted comprising a frit (1)and a fitting (2). In FIG. 1b ) a separator with an upper frit (3) and alower frit (4) and a fitting (2) is shown. In FIG. 1c ) the verticalcross-section of the guide ring of the separator comprising two axiallysymmetric cross-section areas (5 and 6) is shown, wherein each of theaxially symmetric cross-section areas has a) a tapering structure,wherein the tapering is from the outside to the inside of the guidering, and b) a notch (8) with an opening directed to the inside of theguide ring for mounting a frit.

The chromatography column separator as reported herein comprises inaddition to the frit a fitting or guide ring (both terms can be usedinterchangeably) for sealing the distance between the outer edge of thefrit and the chromatography column wall when the separator is placedinside a chromatography column. The fitting or guide ring is in oneembodiment made from a flexible material, such as rubber, plastic,silicone, polytetrafluoroethylene, polyethylene, polypropylene, or thelike. The fitting or guide ring has to be flexible in order to levelsmall differences in the inside diameter of one chromatography column orbetween different chromatography columns of the same nominal diameter toprevent the liquid phase or the chromatography material from passing theseparator outside the frit. It has to be pointed out that the separatoras reported herein can be passed only by the mobile phase but not by thechromatography material. That is the frit has a pore size that issmaller than the size of particles of the chromatography material.

The fitting or guide ring has a circular shape with a cross-section thatcan have any shape as long as it has a rectangular notch for taking upthe frit. For example, in one embodiment the cross-section of thefitting has the shape of a triangle with a rectangular notch for takingup the frit in the corner of the triangle with the biggest inside angle.In a further embodiment the fitting or guide ring has the cross-sectionor provides cross-sectional areas in form of a triangle, in anotherembodiment of a rectangular triangle, wherein the frit is attached tothe corner of the triangle with an inner angle of 90°. In anotherembodiment the cross-section area of the guide ring has a trapezoid fromwith the rectangular notch for taking up the frit being at the shorterside of the parallel sides. In one embodiment the fitting has the formof a rectangle, in another embodiment of a rectangle with inner anglesof 90°, 90°, 80° and 100°. In one embodiment are the inner angles of 80°and 100° are at the upper side or at the bottom side of the rectangle.In one embodiment the longest side of the guide ring is the outer edgeof the separator and has contact to the chromatography column wall whenthe separator is placed inside a chromatography column. In anotherembodiment the longest side has a vertical orientation. In still anotherembodiment the cross-section areas of the fitting or guide ring have theform of a rectangle with inner angles of 90° with the rectangular notchfor taking up the frit in one of the shorter sides or in case of asquare in one of the sides. The side with the notch is the side of thefitting directing to the center of the separator and likewise thechromatography column or in other words the notch is in the side of thefitting or guide ring that is parallel to the flow direction of themobile phase and that has a diameter smaller than the outer diameter ofthe frit. The guide ring has besides the preventing of liquid phase andchromatography material particles passing the separator beside frit thefunction to prevent the canting and therewith stalling of the entireseparator in the chromatography column during the compression andexpansion of the chromatography material upon the applying of theoutside pressure. The separator as reported herein is placed inside thechromatography material packed into a chromatography column. Theseparator can be moved freely and placed exactly inside the column as itcan slide along the inner wall of the chromatography column. This isuseful during the packing of the chromatography column and for removingthe separator from the chromatography column. Thus, in order to preventthe formation of a cavity below the separator and therewith tonegatively impact the chromatography separation process the separator asreported herein is constructed in a way to allow a sliding of theseparator along the inner wall of the chromatography column along withthe compression and expansion of the chromatography material into whichthe separator is embedded. In one embodiment the verticalcross-sectional areas of the guide ring have the form of a triangle ortrapezoid in which the guide ring has a tapering structure, wherein thetapering is from the outside to the inside of the guide ring, that isthe guide ring is at its outer edge higher than at its inner edge or atthe notch, respectively. In one embodiment the outer edge of the guidering has a height that is at least 1.5 times the height of the notch. Inanother embodiment the outer edge of the guide ring has a height that isat least 1.5 times, or two times, or three times, or more than threetimes the height of the notch.

If the separator comprises an upper frit and a lower frit in oneembodiment the fitting or guide ring is a single fitting or ring and inanother embodiment the fitting or guide ring is made of an upper fittingor ring and a lower fitting or ring. In the latter case the two fittingsor rings have in one embodiment a contact-area comprising the lower sideof the upper fitting or ring and the upper side of the lower fitting orring, whereby the contact sides are flat, i.e. have no notch or groove,and are in line with the lower side of the upper frit and the upper sideof the lower frit, i.e. the lower side of the upper fitting and thelower side of the upper frit form a single surface without offset andlikewise the upper side of the lower fitting or ring and the upper sideof the lower frit form a single surface without offset whereby bothsurfaces are parallel.

Generally the smallest inner diameter of the fitting or guide ring or ofthe upper fitting and of the lower fitting is smaller than the outerdiameter of the frit, i.e. the fitting or guide ring extends over theouter perimeter of the frit towards the center of the chromatographycolumn.

In one embodiment the fitting or guide ring comprises at its uppersurface three or more holes each with a screw thread for use with achromatography column separator application device. In a furtherembodiment comprises the lower fitting or guide ring at its uppersurface three or more holes without a screw thread. In one embodimentthe fitting comprises from three to six holes either with or withoutscrew thread.

In FIG. 2 cross-sections areas of different fittings or guide rings areshown. In FIG. 2a ) a cross-section area of a triangular fitting isshown, in FIG. 2b ) a cross-section area of a trapezoid fitting is shownand in FIG. 2c ) a cross-section area of a rectangular fitting is shown.It can be seen from the cross-section areas in FIG. 2 that the fittingsor guide rings extend well over the outer perimeter of the frit.

In FIG. 3 a perspective view of a separator with a guide ring comprisingthree holes with screw thread is shown.

The chromatography column separator can be inserted into thechromatography column with different methods. One method for insertingthe chromatography column separator as reported herein into achromatography column containing a first fraction of a chromatographymaterial comprises placing the chromatography column separator on top ofthe surface of the liquid or chromatography material or chromatographymaterial slurry inside the chromatography column, applying a pressure tothe top of said chromatography column and therewith moving thechromatography column separator along the flow direction of the mobilephase inside the chromatography column until the chromatography columnseparator is at a predetermined position inside the chromatographycolumn.

In a different or alternative method a chromatography column separatorapplication device is used. This device comprises a central, bell-likeshaped corpus, a means for moving the device up and down which isattached to the top of the device, at least three connectors to thechromatography column separator, whereby the connectors areconnected/fixed to the separator and the corpus, and at least one holein the corpus for pressure adjustment between the area below the deviceand the area above the device when placed in a chromatography column. Inone embodiment the connectors are bars with a screw thread at theirlower end for fixing the connectors to the fitting of the chromatographycolumn separator. After having placed the separator at its predeterminedposition the corpus of the device is removed from the column, theconnectors are screwed out of the fitting of the separator and are alsoremoved from the column. Likewise the removal of a separator comprisinga single frit from the column is made by reversing the sequence ofsteps.

If the separator comprises an upper frit and a lower fit the upper fritis removed by reversing the sequence of steps as outlined before. Forremoving the lower frit a grabber is used that hooks in the holespresent in the upper surface of the lower guide ring.

In FIG. 4 a chromatography column separator with a guide ring comprisingthree holes with a screw thread attached via three connectors to achromatography column separator application device is shown.

The packing of a chromatography column with a chromatography materialwith an embedded chromatography column separator as reported herein issplit up into two packing phases. The packing begins with the packing ofa first fraction of the chromatography material into the columnaccording to general procedures. Afterwards the chromatography columnseparator as reported herein is place on top of the first fraction ofthe chromatography material. Finally the second fraction of thechromatography material is packed into the column on top of theseparator according to general procedures. This packing method is apacking from the bottom to the top. In contrast columns not containing aseparator according to the current invention are packed from the toprequiring among other things higher packing pressure. Thus, thechromatography column separator as reported herein provides a means forpacking a chromatography column in two sequential steps if one separatoris used or in three or more sequential steps if two or more separatorsare used. With the separator as reported herein the column is divided inan upper chamber and a lower chamber (one separator) or a lower chamber,a middle chamber, and an upper chamber (two separators) whereof eachitself is equivalent to a chromatography column with reducedchromatography material bed height. With the dividing of thechromatography column in smaller chambers the volume (of thechromatography material in one chamber) to surface (of the chamber)ratio is changed, i.e. lowered, and the stability of the chromatographymaterial packing is increased.

In FIG. 5 chromatography columns comprising one (FIG. 5a ), two (FIG. 5b), and three (FIG. 5c ) chromatography column separators are depicted.

The time point at which a chromatography material has to be replaced hasto be determined individually for each combination of chromatographymaterial, polypeptide to be purified and chromatography conditions. Thiscan be depending e.g. on the specification to be followed or on theyield. Once it was decided that the chromatography material has to bechanged the fraction of the chromatography material in the upper chambercan be removed without disturbing or damaging the packing of thefraction of the chromatography material in the lower chamber of thechromatography column. The break down of the chromatography materialduring the use of the chromatography column results in chromatographymaterial particles with a reduced diameter (size). The smaller theseparticles get the more pores of the frit of the separator are blocked.Therefore, it is also advisable to remove the separator from the column,clean the frit and introduce the separator again into the column oncethe fraction of the chromatography material in the upper chamber hasbeen removed and before new chromatography material is filled into theupper chamber of the chromatography column. If a separator comprising anupper frit and a lower frit is used only the upper frit has to beremoved and the lower frit can remain in place. In this embodiment thepacking of the chromatography material in the lower chamber is less oreven not at all disturbed.

The chromatography column separator as reported herein can be used inany chromatography column in order to divide the chromatography columninto two, three or more individual and independent chambers.

The polypeptide erythropoietin was available in our laboratory at thetime the invention was made in sufficient quantity to evaluate theproperties of the chromatography column separator. This is not intendedto be a limitation of the scope of the invention but only presented asan example to illustrate the current invention.

In FIG. 6 the UV-absorption elution diagram of a chromatography oferythropoietin with a Vydac C4 chromatography material is shown, wherebythe chromatogram a) is obtained with a chromatography column comprisingno chromatography column separator and chromatogram b) is obtained witha chromatography column comprising one chromatography column separatoras reported herein. It can be seen that the chromatograms are identicalthereby showing no influence of the separator on the chromatographicalbehavior. Thus, with the separator according to the invention it ispossible to divide the chromatography material in the chromatographycolumn in two or more distinct fractions or chambers without interferingwith the chromatographical separation process.

In FIG. 7 the increase of the backpressure of a chromatography columncomprising a chromatography column separator as reported herein insuccessive regeneration cycles of a multi-use chromatography column isshown. The regeneration step is best suited to exemplify this as in thisstep the highest backpressure in a chromatography cycle occurs. As canbe seen from FIG. 7 the backpressure continuously increases insuccessive regeneration steps probably due to the increasing destructionof the chromatography material. After cycle 58 the used and broken downchromatography material in the upper chamber of the chromatographycolumn is replaced with fresh, i.e. new, chromatography material. Withthe replacement only of the upper fraction of the chromatographymaterial a dramatic reduction of the backpressure in the followingregeneration step is achieved. Thus, the chromatography column separatoras reported herein firstly does not affect the chromatographicseparation and secondly provides for an easy, efficient and costeffective renewal of a chromatography column packing. It has to bepointed out that the chromatography material in this column was exchangea second time and therewith the chromatography material in the lowerchamber was operated for 98 consecutive separation cycles without theneed to be changed. Without the use of a separator as reported hereinthe column material has to be changed at an average after 15 cycles.

In FIG. 8 the determination of the plate number of a Vydac C4chromatography column is shown. In chromatogram a) the peak of thetracer substance used for the determination of the plate number is splitup into two peaks. This shows that the chromatography column packing hasa defect, e.g. a rupture in the chromatography material e.g. which opensan alternative flow path giving rise to the observed peak doubling. Inchromatogram b) the determination of the plate number after thereplacement of the fraction of the chromatography material in the upperchamber of the chromatography column is shown. No more peak doubling canbe seen.

Furthermore, with the chromatography column separator as reported hereinit is possible to pack a first chromatography material into the lowerchamber and a second, different chromatography material into the upperchamber of a chromatography column. With two different chromatographymaterials a Hybrid-Chromatography column is obtained.

In FIGS. 9 and 10 different UV-absorption elution diagrams of achromatography of an IL13 receptor alpha antibody are shown. Generallyan antibody can be purified by a combination of a cation exchangechromatography and an anion exchange chromatography whereby the ionexchange chromatographies can have any sequence. In FIG. 9 theUV-absorption elution diagram of a first SP-sepharose cation exchangechromatography in a first column (FIG. 9a ) and a second Q-sepharoseanion exchange chromatography in a second column (FIG. 9b ) are shown.In this two step procedure the eluate containing the antibody has beenprocessed prior to the application to the second chromatography column.In FIG. 10 the analytical SEC elution diagram of the purification of theantibody by a Hybrid-Chromatography as reported herein is shown. FIG.10a ) shows the diagram of the analysis of the product peak of FIG. 9b). FIG. 10b ) is the diagram of the product peak of aHybrid-Chromatography with the SP-sepharose in the upper chamber of theHybrid-Chromatography column and the Q-Sepharose in the lower chamber ofthe Hybrid-Chromatography column. FIG. 10c ) provides the diagram of theproduct peak of a Hybrid-Chromatography with the Q-sepharose in theupper chamber of the Hybrid-Chromatography column and the SP-Sepharosein the lower chamber of the Hybrid-Chromatography column. As can be seenfrom the chromatograms presented in FIG. 10 the Hybrid-Chromatographyresults are essentially identical to the separate chromatography stepswith only slightly different retention times due to the differentsequence of the chromatographic steps.

The chromatography column separator as reported herein:

-   -   provides a means for using different types of the same        chromatography material in the upper and lower chromatography        column chambers, e.g. in the upper chromatography column chamber        a chromatography material with higher mechanical stability can        be used and in the lower chromatography column chamber a        chromatography material with lower mechanical stability can be        used;    -   provides a means for replacing used or torn or collapsed        chromatography material in the upper chromatography column        chamber without the need to replace the chromatography material        in the lower chromatography column chamber;    -   provides a means for using in each of the chromatography column        chambers a different chromatography material with different        chromatographical functional groups allowing for the combination        of two different chromatographic methods.

By using the chromatography column separator as reported herein e.g. apressure sensitive chromatography material can be used in the lowerchromatography column chamber as the separator reduces the pressuredirected to the chromatography material in the lower chromatographycolumn chamber. That is by using a separator as reported herein apressure reduction inside the chromatography material can be effected.Thus, chromatography columns comprising a separator as reported hereinhave a reduced backpressure compared to chromatography columns with thesame bed height but without a separator as reported herein (thebackpressure is the pressure required to force the mobile phase throughthe chromatography material), i.e. the total pressure as well as thepressure changes as well as the pressure increase are reduced. Asoutlined above a reduced pressure and reduced pressure changes canprovide for an increased number of chromatography cycles without therequirement to change the chromatography material inside thechromatography column. In one embodiment the chromatography material inthe lower chromatography column chamber of a chromatography columncomprising a chromatography column separator with distance holders isselected from DEAE-sepharose or HA-Ultrogel.

Additionally by using a separator as reported herein a chromatographycolumn can be operated at increased flow rates of the mobile phase. Thisis due to the reduction of the required pressure which has to be appliedto the column for achieving a predetermined flow rate (see FIG. 11).

In one embodiment the chromatography column separator comprises distanceholders directed to the lower end of the chromatography column. Inanother embodiment the chromatography column separator comprises threeto six distance holders.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Exemplary chromatography column separators according to theinvention: a) separator with a single frit comprising a frit (1) and afitting (2); b) separator with an upper frit (1) and a lower frit (3)and an upper fitting (2) and a lower fitting (4); c) verticalcross-section of the guide ring of the separator comprising two axiallysymmetric cross-section areas (5 and 6) each having i) a taperingstructure, wherein the tapering is from the outside to the inside of theguide ring, and i) a notch (8) with an opening directed to the inside ofthe guide ring for mounting a frit.

FIG. 2 Cross-section areas of different fittings: a) cross-section of atriangular fitting, b) cross-section area of a trapezoid fitting, c)cross-section area of a rectangular fitting, d) cross-section area of anupper fitting and a lower fitting.

FIG. 3 Perspective views of a fitting comprising three holes with screwthread are shown.

FIG. 4 Cross-section of a chromatography column separator with a fittingcomprising three holes with a screw thread attached via three connectorsto a chromatography column separator application device.

FIG. 5 Chromatography columns comprising one (a), two (b), and three (c)chromatography column separators.

FIG. 6 UV-absorption elution diagram of a chromatography oferythropoietin with a Vydac C4 chromatography material, whereby thechromatogram a) is obtained with a chromatography column comprising nochromatography column separator according as reported herein andchromatogram b) is obtained with a chromatography column comprising onechromatography column separator as reported herein.

FIG. 7 Increase of the backpressure of a chromatography columncomprising a chromatography column separator as reported herein insuccessive regeneration cycles of a multi-use chromatography column.

FIG. 8 Determination of the plate number of a Vydac C4 chromatographycolumn.

FIG. 9 UV-absorption Q-sepharose elution diagram of a chromatogram of anIL13 receptor alpha antibody: a) SP-sepharose cation exchangechromatography; b) Q-sepharose anion exchange chromatography.

FIG. 10 Analytical SEC elution diagram: a) diagram of the analysis ofthe product peak of FIG. 9; b) diagram of the product peak of aHybrid-Chromatography with the SP-sepharose in the upper chamber of theHybrid-Chromatography column and the Q-Sepharose in the lower chamber ofthe Hybrid-Chromatography column; c) diagram of the product peak of aHybrid-Chromatography with the Q-sepharose in the upper chamber of theHybrid-Chromatography column and the SP-Sepharose in the lower chamberof the Hybrid-Chromatography column.

FIG. 11 Diagram showing resulting column (back) pressure vs. set mobilephase flow for an anion exchange chromatography material (DEAESepharose). Diamonds: chromatography column without a separator asreported herein; triangles: chromatography column with separator asreported herein. It can be seen that a reduction of back-pressure forhigher bed heights can be achieved with a separator as reported herein.

EXAMPLE 1 Fermentation and Purification of Erythropoietin

The Fermentation and purification of Erythropoietin was carried out asreported in European patent No. 1 064 951 B1. The data presented hereinwere obtained in the reversed phase HPLC on a Vydac C4 chromatographymaterial as reported in Example 1d) of EP 1 064 951.

The RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles,the surfaces of which carry C4-alkyl chains. The separation ofErythropoietin from the proteinaceous impurities is based on differencesin the strength of hydrophobic interactions. Elution is performed withan acetonitrile gradient in diluted trifluoroacetic acid. PreparativeHPLC is performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silica gel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoro-acetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer.

EXAMPLE 2 Production of an Anti-(IL-13Rα1) Antibody

An anti-IL13 receptor alpha antibody was produced according to the dataand methods reported in WO 2006/072564, especially in accordance withExamples 10 to 12.

Sequential-Chromatography

For the ion exchange chromatography the protein A eluate comprising theanti-IL13R alpha antibody is adjusted to pH 6.5 and applied to aSP-Sepharose cation exchange chromatography column that has beenequilibrated with 10 mM potassium phosphate buffer. After a wash stepwith 10 mM potassium phosphate buffer the antibody is eluted with a 50mM potassium phosphate buffer pH 6.5. The pH value of the SP-Sepharoseeluate is adjusted to pH 7.1 and applied to a Q-Sepharose anion exchangechromatography column that has been equilibrated with 35 mM potassiumphosphate buffer. The purified antibody is obtained from the flow-thoughof the anion exchange chromatography column.

Hybrid-Chromatography

For the Hybrid-ion exchange chromatography with a chromatography columncontaining a chromatography column separator according to the currentinvention the protein A eluate comprising the anti-IL13R alpha antibodyis adjusted to pH 7.1 and applied to the Hybrid-Chromatography columnthat has been equilibrated with 10 mM potassium phosphate buffer. Aftera wash step with 10 mM potassium phosphate buffer the antibody is elutedwith a 20 mM potassium phosphate buffer pH 7.1.

The invention claimed is:
 1. A chromatography column comprising aseparator comprising an O-shaped guide ring having a verticalcross-section comprising two separate axially symmetric cross-sectionareas, wherein each of the axially symmetric cross-section areas has i)a tapering structure, wherein the tapering is from the outside to theinside of the guide ring, and ii) a notch with an opening directed tothe inside of the guide ring for mounting a frit, wherein the notch is arectangular notch, wherein further each of the cross-section areas has atriangular shape and the longest side has a length of at least 1.5 timesthe diameter of the notch, and a frit mounted into the guide ring;wherein the separator is slidably disposed inside a chromatographycolumn such that the separator is freely movable within thechromatography column by sliding along the inner wall of thechromatography column; wherein an outside of the guide ring is incontact with the inner wall of the chromatography column.
 2. Thechromatography column of claim 1, wherein the frit is selected from thegroup consisting of a) a single frit, or b) two frits with an upper fritand a lower frit.
 3. The chromatography column of claim 2, wherein a)the single frit has a pore size of from 1 μm to 20 μm or b) each of thetwo frits has a pore size of from 1 μm to 20 μm independently of eachother whereby the pore size of the upper frit is smaller than the poresize of the lower frit.
 4. The chromatography column of claim 1, whereinthe frit is made of metal, silicone, polypropylene, polyethylene,polytetrafluoroethylene, sintered materials or combinations thereof. 5.The chromatography column of claim 4, wherein the separator comprisesdistance holders all attached to one side of the separator; wherein theseparator has a shape.
 6. A chromatography column comprising more thanone chromatography column separators according to claim 1, comprisingmore than one separator.
 7. The chromatography column of claim 6 whereinat least one of the chromatography column separators is in contact witha first chromatography material and in contact with a secondchromatography material, whereby the first and the second chromatographymaterial are a) chromatography materials with the same chromatographicalfunctional group and of the same or different particle size, or b)chromatography materials with different chromatographical functionalgroups.
 8. The chromatography column of claim 1, wherein the separatorseparates the chromatography column into an upper chromatography columnchamber and a lower chromatography column chamber.